Cartilage is an essential tissue in developing and adult humans. Sadly, this is best illustrated by the prevalence and severity of birth defects due to cartilage malformations, growth retardations due to growth plate defects in children, and articular cartilage degeneration diseases in adults. Today, however, no suitable treatments exist for most of these diseases, and this is largely due to incomplete understanding of the mechanisms underlying chondrogenesis. The main goal of this project is to help lift this barrier by increasing knowledge of the action and regulation of Sox9, a master transcription factor in chondrogenesis. Sox9 is well known to activate the early chondrocyte differentiation program, and our recent data reveal that it remains a master transcription factor in the growth plate throughout chondrocyte hypertrophy. It sustains columnar chondrocyte proliferation, delays prehypertrophy, and ensures hypertrophy. Based on these data, we propose that important mechanisms must exist to determine the specific activity of Sox9 at each chondrocyte developmental stage from early to hypertrophic differentiation. Aim 1 is to test the hypothesis that the redundant proteins Sox5 and Sox6 boost the ability of Sox9 to activate the differentiation program of early chondrocytes by increasing the efficiency of Sox9 binding to gene enhancers. Aim 2 is to test the hypothesis that Sox9 phosphorylation by the cAMP-dependent protein kinase A critically contributes to delaying chondrocyte maturation downstream of parathyroid hormone-related protein signaling. Aim 3 is to test the hypothesis that specific transcriptional mechanisms allow Sox9 to activate the hypertrophic chondrocyte program and most specifically the Col10a1 gene. All aims will be reached using genetic approaches in the developing mouse and state-of-the-art genomics, cellular and molecular approaches in vitro. We anticipate that the achievement of this project will lead to a deeper knowledge of the mechanisms that govern the multi-step differentiation pathway of chondrocytes. This knowledge will provide novel insights into mechanisms underlying cartilage malformation and degeneration diseases and will help find suitable treatments.